The production of the enzyme manganese human superoxide dismutase (hSODm) by recombinant DNA techniques is described. Both natural and modified enzymes are produced utilizing novel DNA constructs, plasmids and transformed microbial expression systems.
Superoxide dismutase (xe2x80x9cSODxe2x80x9d) is in fact a variety of different enzymes found in most living organisms. One function in mammals is to destroy superoxide. Superoxide is a material naturally produced during phagocytosis and aerobic metabolism. The superoxide dismutases are characterized in families based on the metal ion associated with the enzyme, where the ions can be iron, manganese, copper, and copper and zinc. Superoxide dismutase, e.g., from bovine liver, has found clinical use, particularly as an anti-inflammatory agent in mammals including humans and to decrease tissue injury due to reperfusion (post-ischemic). Other utilities include scavenging superoxide anions due to exposure of a host to various superoxide-inducing agents, e.g. radiation, paraquat, etc.; prophylaxis or therapy for certain degenerative diseases, e.g., emphysema; food preservation; and the like.
It is therefore important that stable supplies of physiologically acceptable superoxide dismutase be made available, particularly for use in vivo as an anti-inflammatory agent or for other therapeutic purposes. For human application it would be preferable to employ the homologous enzyme to prevent or minimize possible immune response. By employing recombinant DNA techniques, there is the opportunity to produce products efficiently, which have the desired biological activities of superoxide dismutase, such as immunological and enzymatic activities.
The primary structure of human liver manganese superoxide dismutase was described by Barra et al., J. B. C., 259:12595-12601, (1984). Either bacterial iron superoxide dismutase (FeSOD) or bacterial manganese superoxide dismutase (MnSOD) were shown to be required as a defense against oxygen toxicity by Carlioz et al., EMBO Journal, 5:623-630, (1986). The amino-terminal processing of methionine by yeast was shown by Tsunasawa et al., J.B.C., 260:5382-5391, (1985). Human copper/zinc superoxide dismutase was described by Hallewell, et al., Nucleic Acids Research, 13:2017-2034, (1985). A superoxide dismutase produced in Serratia marcescens is described in EPO application 0172577. An immobilized superoxide dismutase was described in EPO application 070656.
The amino acid sequence of human erythrocyte Cuxe2x80x94Zn superoxide dismutase was described in Jabusch et al., Biochemistry (1980) 19:2310-2316 and Barra et al., FEBS Letters (1980) 120:53-55. Bovine erythrocyte Cuxe2x80x94Zn SOD was described by Steinman et al., J. Biol. Chem. (1974) 249:7326-7338. A SOD-1 (Cuxe2x80x94Zn SOD) cDNA clone is described by Lieman-Hurwitz et al., Proc. Natl. Acad. Sci. USA (1982) 79:2808-2811.
Novel compositions are provided comprising nucleic acid sequences for the expression of polypeptides exhibiting the biological properties of human manganese superoxide dismutase (xe2x80x9chSODmxe2x80x9d). Also provided are methods for producing such polypeptides employing recombinant DNA techniques and microorganism hosts. The subject polypeptides find use in vivo and in vitro in destroying superoxide.
Also provided is a novel modified hSODm. A modified DNA coding for hSODm with the first two amino acids, lysine and histidine, removed, resulted in a polypeptide with the third amino acid, serine, positioned adjacent to the translation initiating amino acid methionine. This modified hSODm permitted removal of the amino terminal methionine by processing enzymes.
A method of treating a patient having inflammatory joint disease or post-ischemic tissue injury is also provided.